PROJECT SUMMARY Invasive fungal infections caused by a variety of fungal pathogens are on the rise in the United States and globally. Alarmingly, a growing number of these clinical isolates are resistant to anti-fungal treatments. There have been recent calls by the CDC and NIH for the development of novel strategies to develop diagnostics and therapeutics to combat fungal infections. Invasive infections of the respiratory system by the clinically relevant fungus Aspergillus fumigatus occur in a wide variety of immunosuppressed populations and can be associated with very high mortality rates. Strains of A. fumigatus resistant to common anti-fungals are also on the rise. This study aims to dissect a component of the host immune system (known commonly as Nlrx1) that is capable of amplifying the recruitment of important white blood cell populations (neutrophils), and regulating killing of fungal conidia and hyphae by airway epithelial cells (the first cells in contact with inhaled fungi). The applicant has shown that loss of Nlrx1 results in increased fungal growth as well as host mortality suggesting its function is critical for immune signaling as well as cell specific defense responses. How Nlrx1 regulates these processes is unknown. The preliminary data suggests this process occurs through non-oxidative mechanisms. Specific Aim 1 is designed to identify anti-fungal proteins that are generated and secreted by airway epithelial in a manner dependent upon Nlrx1. The expression of these proteins is thought to be mitigated in Nlrx1 deficient cells, which is a reasonable explanation for the enhanced fungal germination and growth observed in vivo and in vitro. Completion of Aim 1 will provide the identification and characterization of novel host derived anti-fungal proteins that reduce fungal germination and hyphae growth in vitro and in vivo. Aim 2 is designed to determine if expression of Nlrx1 in lung tissue, mediates enhanced fungal growth and immune signaling observed in whole body knockouts. This will be done through generation of lung specific knockouts of Nlrx1 via available Cre-lox mice. Aim 2 will also provide mechanistic insight into how Nlrx1interacts with different cell membrane receptors that sense fungi. Completion of this proposal will provide novel insight into how lung tissue Nlrx1 regulates both oxidative and non-oxidative defense mechanisms in response to conidia and hyphae.